Injectable microspheres for dermal augmentation and tissue bulking

ABSTRACT

The present invention relates to elastic, hydrophilic and substantially spherical microspheres useful for dermal augmentation and tissue bulking. The invention provides injectable compositions comprising the microspheres and a biocompatible carrier for use in dermal augmentation. The present invention further provides methods of dermal augmentation and tissue bulking, particularly for the treatment of skin contour deficiencies, Gastro-esophageal reflux disease, urinary incontinence, and urinary reflux disease, using the injectable compositions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/704,919 filedNov. 12, 2003 (abandoned), which is a continuation of U.S. Ser. No.09/528,991 filed Mar. 20, 2000 (now U.S. Pat. No. 6,660,301), which is acontinuation-in-part U.S. Ser. No. 09/263,773; filed Mar. 5, 1999 (nowU.S. Pat. No. 6,335,028), which claims the benefit of U.S. ProvisionalSer. No. 60/077,166 filed Mar. 6, 1998, each of which is herebyincorporated by reference in its entirety.

FIELD

The present invention relates to dermal augmentation and tissue bulking,particularly for the treatment of gastroesophageal reflux disease,urinary incontinence, urinary reflux disease, or skin contourdeficiencies and wrinkles, using injectable microspheres.

BACKGROUND Gastroesophageal Reflux Disease (“GERD”)

Although gastroesophageal reflux is a normal physiological phenomenon,in some cases it is a pathophysiological situation that can result in avariety of symptoms which may become severe in extreme cases.Gastro-Esophageal Reflux Disease (“GERD”), describes a backflow ofacidic and enzymatic liquid from the stomach to the esophagus. It causesburning sensations behind the sternum that may be accompanied byregurgitation of gastric acid into the mouth or even the lung.Complications of GERD which define the severity of the disease includeesophageal tissue erosion, and esophageal ulcer wherein normalepithelium is replaced by a pathological tissue.

Statistical data indicate that about 35% of the American populationsuffer from heartburn at least once a month and between 5 to 10% once aday. More importantly for this kind of disease about 2% of the Americanpopulation suffer from GERD based on medical evidence data fromendoscopic examination. This disease is related to the age ofindividuals and seems to increase after 40 years of age. (Nebel O. T. etal., Am. J. Dig. Dis., 21(11):953-956 (1976)).

In normal patients, after a meal the lower esophageal sphincter remainsclosed, but in patients with GERD, it relaxes and allows some acidicmaterial to reflux into the esophageal tube as a result of stomachcontractions. Actually GERD can be attributed primarily to transientrelaxation of the lower esophageal sphincter. In other cases, GERD canbe attributed to decreased resting tone of the lower esophagealsphincter or to congenital small dimension of the sphincter itself.Other causes also exist which contribute to varying degrees to theexistence and severity of this disease.

In addition, there are external factors that contribute to exacerbatethe symptoms of GERD, which conditions include eating fatty foods,caffeine intake, smoking, tight clothing and certain medications.Decrease in salivation can also be a factor that exacerbates GERD, sinceunder normal conditions saliva, which is an alkaline liquid, aids inneutralizing acidic reflux and therefore diminishing the duration of theacidic exposure of the esophagus.

Erythema is one of the first visible signs of GERD, which can be seen byendoscopy. Tissue erosion indicates more advanced disease which can thenbecome deep ulcers and lead to cancer (adenocarcinoma increases inincidence faster than other types of cancer). Diffuse ulceration andspecific complications occur in about 3.5% of patients less than 65years of age with esophageal obstruction, blood loss, and in some cases,perforation. Ulcerative situations not only lead to complications, butthey are also more resistant to treatments. Although severecomplications are uncommon in young patients, they occur in about 20-30%of patients over 65 (Reynolds J. C, Am. J. Health-Sys. Pharm 53,(1996)).

Prior to the present invention, in an attempt to increase the functionof the sphincter, bulking methods using bovine collagen and Teflon pastehave been used in patients. Both methods have been unsuccessful,however, as these materials migrate over time from the initial site ofimplantation.

At present, GERD is generally managed by over-the-counter (“OTC”)antacids or prescription drugs, including proton pump inhibitors,motility agents and H₂ blockers. In addition, a portion of GERD patientsrequire surgical intervention; the most common type of surgery isfundoplication which can be done by conventional surgical techniques, orusing laparoscopic techniques. However, fundoplication surgery carriesthe risk of serious side effects and is only marginally successful incuring GERD. Respiratory symptoms are also associated with GERD in about50% of patients, and in patients undergoing fundoplication, theserespiratory symptoms can even increase (76% reported in a study byJohnson W. E. et al., Archives of Surgery, 131:489-492 (1996)).

Urinary Incontinence and Urinary Reflux Disease

Urinary incontinence is a prevalent problem that affects people of allages and levels of physical health, both in the community at large andin healthcare settings. Medically, urinary incontinence predisposes apatient to urinary tract infections, pressure ulcers, perineal rashes,and urosepsis. Socially and psychologically, urinary incontinence isassociated with embarrassment, social stigmatization, depression, andespecially for the elderly, an increased risk of institutionalization(Herzo et al., Ann. Rev. Gerontol. Geriatrics, 9:74 (1989)).Economically, the costs are astounding; in the United States alone, overten billion dollars per year is spent managing incontinence.

Incontinence can be attributed to genuine urinary stress (urethrahypermobility), to intrinsic sphincter deficiency (“ISD”), or both. Itis especially prevalent in women, and to a lesser extent incontinence ispresent in children (in particular, ISD), and in men following radicalprostatectomy.

One approach for treatment of urinary incontinence involvesadministration of drugs with bladder relaxant properties, withanticholinergic medications representing the mainstay of such drugs. Forexample, anticholinergics such as propantheline bromide, and combinationsmooth muscle relaxant/anticholinergics such as racemic oxybutynin anddicyclomin, have been used to treat urge incontinence. (See, e.g., WeinA. J., Urol. Clin. N. Am., 22:557 (1995)). Often, however, such drugtherapies do not achieve complete success with all classes ofincontinent patients, and often results in the patient experiencingsignificant side effects.

Besides drug therapies, other options used by the skilled artisan priorto the present invention include the use of artificial sphincters (LimaS. V. C. et al., J. Urology, 156:622-624 (1996), Levesque P. E. et al.,J. Urology, 156:625-628 (1996)), bladder neck support prosthesis (KondoA. et al., J. Urology, 157:824-827 (1996)), injection of crosslinkedcollagen (Berman C. J. et al., J. Urology, 157:122-124 (1997), Perez L.M. et al., J. Urology, 156:633-636 (1996); Leonard M. P. et al., J.Urology, 156:637-640 (1996)), and injection of polytetrafluoroethylene(Perez L. M. et al., J. Urology, 156:633-636 (1996)).

A recent well known approach for the treatment of urinary incontinenceassociated with ISD is to subject the patient to periurethral endoscopiccollagen injections. This augments the bladder muscle in an effort toreduce the likelihood of bladder leakage or stress incontinence.

Existing solutions to circumvent incontinence have well known drawbacks.The use of artificial sphincters for children with intractableincontinence requires long term surveillance of the urinary tractbecause of the potential for renal failure after device placement(Levesque P. E. et al., J. Urology, 156:625-628 (1996)). Whileendoscopically directed injections of collagen around the bladder neckhas a quite high success rate in sphincter deficiency with nosignificant morbidity, the use of collagen can result in failures thatoccur after an average of two years and considerations need to be givento its cost effectiveness (Khullar V. et al., British J. Obstetrics &Gynecology, 104:96-99 (1996)). In addition, deterioration of patientcontinency, probably due to the migration phenomena (Perez L. M. et al.)may require repeated injections in order to restore continency(Herschorn S. et al., J. Urology, 156:1305-1309 (1996)).

The results with using collagen following radical prostatectomy for thetreatment of stress urinary incontinence have also been generallydisappointing (Klutke C. G. et al., J. Urology, 156:1703-1706 (1996)).Moreover, one study provides evidence that the injection of bovinedermal collagen produced specific antibodies of IgG and IgA class.(McCell and, M. and Delustro, F., J. Urology 155, 2068-2073 (1996)).Thus, possible patient sensitization to the collagen could be expectedover the time.

Despite of the limited success rate, transurethral collagen injectiontherapy remains an acceptable treatment for intrinsic sphincterdeficiency, due to the lack other suitable alternatives.

Urinary reflux disease, or “vesicoureteral reflux” in its medical term,simply means that urine goes backwards in the ureters during urination.The disease often occurs in young children. The ureter is the tube whichconnects the kidneys with the bladder. Urine is supposed to go in onedirection: from the kidneys to the bladder. When urine goes up from thebladder to the kidneys, it can result in health problems for the person.

Urinary reflux can lead to kidney damage. Refluxing urine can carrybacteria to the kidney, where it can cause kidney infection. Childrenwith reflux of urine are much more likely to have kidney infection thanchildren who do not have reflux. The combination of reflux and infectioncan lead to areas of permanent kidney damage or “renal scarring.” Thisscarring is detected by doing an X-ray called an intravenous pyelogram(IVP), or preferably, a renal scan. If it is extensive enough, thescarring can lead to loss of function of one or both kidneys.

The key to preventing renal scarring is preventing kidney infections.This is currently being carried out in two ways. In most cases, longterm prophylactic antibiotics are given. The other method of preventingurinary tract infections is surgical correction of the reflux. Bothmethods, however, have drawbacks. Long term use of antibiotics may causeunpredictable side effects and surgical procedures involve unnecessaryrisks.

Even though many urinary reflux disease will go away on its own inchildren, some cases often lead to severe kidney and urinary tractinfections and even total kidney failure. There is a need, therefore,for a safe, effective, less intrusive, and long lasting method oftreating urinary reflux disease.

Skin Deficiencies

Damage to the skin due to aging, environmental exposure to the sun andother elements, weight loss, child bearing, disease such as acne andcancer, and surgery often results in skin contour deficiencies and otherskin anomalies. In order to correct contour deficiencies and otheranomalies of the skin, people often resort to cosmetic surgery, such asface lifts and skin tucks. Cosmetic surgery, however, has severaldrawbacks, in addition to the high cost associated with it. It isusually an invasive and risky procedure, having the potential of leavingscars in areas of operation and affecting normal biological andphysiological functions. Furthermore, cosmetic surgery is often alimited option, available only for certain skin deficiencies.

In addition to cosmetic surgery, various other methods are used toremove or ameliorate the deficiencies with different levels of success.The use of injectable material for soft tissue augmentation is a methodoften used. The advantage of using hypodermic needles as a deliverydevice for dermal augmentation reflects the advantages of usinghypodermic needles in general: easy, precise and, usually, non-invasivedeliveries. Yet, the requirement for such use is also quite strict: thematerial to be delivered must be deliverable through the needles, whichmeans the material must be able to easily pass through the hollowcenters of the needles.

One method of dermal augmentation using injectable material is liquid orsemi-liquid injections, usually containing collagen. The best knownexample is a collagen preparation manufactured by Collagen Corporation(now part of Inamed Corporation) and marketed by C. R. Bard. However,collagen is a naturally occurring substance which the body mayenzymatically degrade and eliminate over time, thus requiring repeattreatments. Also, collagen may be displaced within the tissue in whichit was originally injected, thereby reducing or eliminating the intendeddermal augmentation effect. Collagen is also digested directly(biochemically), through macrophages, through the lymphatic system, orby other means. Even more alarming from a cosmetic perspective, collagenmay move from the initial site of injection, causing unsightly bumps andbulges under the skin at undesired locations. See, e.g., Millikan, LongTerm Safety and Efficacy with Fibrel in the Treatment of CutaneousScars, J Dermatol Surg Oncol, 15:837-846 (1989).

Injection of liquid silicone has also been used extensively to treatskin deficiencies. However, due to long term side effects, such asnodules, recurring cellulitis, and skin ulcers, the use of injectablesilicone is on the decline. See, e.g., Edgerton et al., Indications forand pitfalls of soft tissue augmentation with liquid silicone, Plast.Reconstr. Surg, 58:157-163 (1976).

MicroParticles

Prior to the present invention, microspheres have been manufactured andmarketed for in vitro use in anchorage dependent cell culture. (VanVezel, A. L., Nature, 216:64-65 (1967); Levine et al., Somatic CellGenetics, 3:149-155 (1977); Obrenovitch et al., Biol. Cell., 46:249-256(1983)). They have also been used in vivo to occlude blood vessels inthe treatment of arteriovascular malformation, fistulas and tumors (See,U.S. Pat. No. 5,635,215, issued Jun. 3, 1997 to Boschetti et al.;Laurent et al., J. Am. Soc. Neuroiol, 17:533-540 (1996); and Beaujeux etal. J. Am. Soc. Neuroial, A:533-540 (1996)).

Further, direct implantation of cells into living tissues such as brainor liver to correct specific deficiencies has been attempted albeit witha number of failures. The major problems associated with direct celltransplantation are the long term viability of the cell transplant andthe immunopathological as well as histological responses. Microparticleswith cells attached on their surface have been used in some in vivoapplications. Cherkesey et al., IBRO, 657-664 (1996), described theculture of adrenal cells on coated dextran beads and the implantationinto mammalian brain to supplant some specific disorders related to6-hydroxydopamine-induced unilateral lesions of the substantia nigra.The pre-attachment of cells to dextran microcarriers allowed forimproved functions of the cells implanted into the brain. Also livercells transplantation has been used to manage acute liver failure, orfor the replacement of specific deficient functions such as conjugationof bilirubin or synthesis of albumin. For this purpose, an intrasplenicinjection of hepatocytes grown on the surface of microspheres wasperformed (Roy Chowdhury et al., in: Advanced Research on Animal CellTechnology, AOA Miller ed., 315-327, Kluers Acad. Press, 1989).

Most of cell implant results have been, however, largely disappointingfor the designated functions (or have had low levels of biologicalfunction).

Prior to the present invention, solid microparticles have also been usedfor the correction of skin deficiencies and for tissue bulking. Forexample, carbon particles, silicone particles, TEFLON(polytetrafluoroethylene) paste, collagen beads andpolymethylmethacrylate spheres, have been used with disappointingresults due to, inter alia, adverse tissue reactions, biologicaldegradation and migration from the initial implantation location.

The problems associated with rigid and non-deformable particles, such ascarbon particles and silicone particles, in tissue bulking or treatingskin deficiencies are that they are either too fragile or too large tobe injected, or too small and are digested or eliminated by the body.Therefore, such particles all have one or more of the followinglimitations: (i) too large to be injected through a 30 gauge or smallerneedle; (ii) particles of irregular shape clump together, makinginjection difficult; (iii) particles are too fragile, resulting inbreakage during injection and digestion of the residues; (iv) injectedparticles are too small and are digested by macrophages or othercomponents of the lymphatic system; and (v) injected particles aredisplaced as they do not adhere to the surrounding cells.

Injectable deformable particles, such as TEFLON(polytetrafluoroethylene) particles, have also been used for tissuebulking and for treating skin deficiencies. However, TEFLON(polytetrafluoroethylene) particles have one or more of the followinglimitations: (1) the particles slide with the tissue and do not stay inplace of injection; (2) the particles deform during and after injection,reducing the intended tissue bulking effect; and (3) the particles aredigested or eliminated by the lymphatic system partly due to the factthat their diameters become smaller as a result of injection.

Therefore, there is a great need for safe, biocompatible, stable andeffective methods of tissue bulking for the treatment of GERD, urinaryincontinence, and urinary reflux disease and methods of dermalaugmentation for treatment of skin disorders.

SUMMARY

The present invention encompasses the use of implantable microparticles,or microspheres or microbeads, in the treatment of GERD, urinaryincontinence, urinary reflux disease and skin deficiencies such as skinwrinkles. In each use the particles are implanted into the appropriatetissue, muscle, organ etc. as a bulking or augmentation agent.

In a preferred embodiment, the invention provides a method of dermalaugmentation, suitable for the treatment of skin deficiencies, and amethod of tissue bulking, suitable for the treatment of GERD, urinaryincontinence, or urinary reflux disease, wherein the microspheres usedare injectable through needles of about 18 gauge or smaller, dependingon the particular method and treatment, and are not capable of beingdigested or eliminated through the lymphatic or the immune system. Thus,the invention encompasses injectable compositions and methods for dermalaugmentation or tissue bulking by injecting using syringes, catheters,needles or other means of injecting or infusing microspheres in a liquidmedium so as to avoid surgical intervention.

The microparticles of the invention, whether implantable by injection orotherwise, are preferably pre-coated, with autologous cells, forexample, muscle cells, fat cells and the like. The microparticles of theinvention are biocompatible non-toxic polymers coated with, linked to orfilled with cell adhesion promoters. The microparticles preferablycontain a positive charge on their surface by way of a cationic monomeror polymer.

In one embodiment, the invention encompasses the treatment ofgastroesophageal reflux disease in a human which comprises implantinghydrophilic biocompatible microparticles comprising (a) a positivecharge and a cell adhesion promoter; and (b) autologous cells layered onthe surface of said beads, into the lower esophageal sphincter. Themicroparticles are preferably microspheres or microbeads which aredescribed in detail herein. The autologous cells are preferably takenfrom the area where the implantation is to be made. Serum or whole bloodtaken from the patient can be used to wash the microparticles prior toimplantation. For GERD treatment, implantation is preferably made byusing standard techniques known to the ordinary skilled artisan, such asinjection (or injections) via syringe or other suitable devices.

In yet another embodiment, the invention encompasses the treatment ofurinary incontinence in a human which comprises implanting hydrophilicbiocompatible microparticles comprising (a) a positive charge and a celladhesion promoter; and (b) autologous cells layered on the surface ofthe beads, into the urinary sphincter. The microparticles are preferablymicrospheres or microbeads as described herein. Further, the autologouscells are preferably taken from the area where the implantation is to bemade. Serum or whole blood from the patient can be used to wash themicroparticles prior to implantation. Implantation is preferably madeusing a syringe or other device suitable for the particular tissue ofimplantation.

In another embodiment, the invention encompasses a method of treatingskin wrinkles in a human which comprises the administration orimplantation of microparticles comprising a hydrophilic copolymer havinga positive charge, and a cell adhesion promoter, which microparticleshave been pre-treated with autologous cells. The microparticles can besimply exposed to the autologous cells or mixed thoroughly withautologous cells prior to implantation. The microspheres are preferablyinjected via syringe or other suitable device through a needle of 30gauge or smaller into the area, or under the area, of the skindeficiencies.

In yet another embodiment, the invention encompasses the treatment oramelioration of skin wrinkles which comprises administering hydrophilicbiocompatible microparticles comprising: (a) a positive charge and acell adhesion promoter; and (b) autologous cells, collagen, collagenderivatives or glucosaminoglycans layered on the surface of the beads,into the area of or surrounding the skin wrinkles. In other words,microspheres or microbeads coated with a cell adhesion promoter andpre-treated with the appropriate tissue bulking cells, are administeredto the area of treatment.

The present invention additionally provides methods of dermalaugmentation and treatment of skin deficiency. Specifically, theinvention provides a method of causing dermal augmentation in a mammalby administering a composition of elastic, hydrophilic, andsubstantially spherical microspheres in a biocompatible carrier to themammal. The composition is injectable through a needle of about 30 gaugeor smaller and the microspheres are not capable of being digested oreliminated by macrophage or other elements of the mammal's immunesystem. According to the present invention, a preferred method ofadministration is injecting the composition into an area of the subjectmammal that is in need of dermal augmentation. A more preferred methodof administration is injecting the composition into the subcutaneouslayer of the subject mammal.

The dermal augmentation method of the present invention is especiallysuitable for the treatment of skin contour deficiencies, which are oftencaused by aging, environmental exposure, weight loss, child bearing,injury, surgery, in addition to diseases such as acne and cancer.Suitable for the treatment by the present invention's method are contourdeficiencies such as frown lines, worry lines, wrinkles, crow's feet,marionette lines, stretch marks, and internal or external scars resultedfrom injury, wound, bite, surgery, or accident.

The invention also encompasses the use of the injectable compositions totreat skin deficiencies caused by diseases such as acne and cancer.

The present invention further provides a method of causing dermalaugmentation in a mammal by administering the injectable suspensionextracorporeally into organs, components of organs, or tissues prior tothe inclusion of said tissues, organs, or components of organs into thebody.

The invention further encompasses method for tissue bulking in a mammalby administering a composition of elastic, hydrophilic, non-toxic andsubstantially spherical microspheres in a biocompatible carrier to themammal. The composition is injectable through needles of about 18 toabout 26 gauge, preferably, 22 to 24 gauge, and preferably administeredby injection directly into the site of treatment, e.g., the sphincter.

Thus, in one embodiment, the tissue bulking method is used for thetreatment of gastroesophageal reflux disease in a mammal; preferably bydirect administration via injection of the composition into the loweresophageal sphincter or the diaphragm of the mammal.

Similarly, the tissue bulking method is used for the treatment ofurinary incontinence or urinary reflux disease via administration of thecomposition into the bladder sphincter or the urethra of the mammal.

The present invention further provides kit for performing dermalaugmentation or tissue bulking. The kit comprises a syringe and a 30gauge or smaller needle for dermal augmentation and an 18 to 26 gaugeneedle for tissue bulking. The syringe optionally comprises acomposition of elastic, hydrophilic, non-toxic and substantiallyspherical microspheres in a biocompatible carrier. Alternatively, thesyringe does not contain a solution or suspension but is accompanied by(a) dry sterilized microspheres which are ready for preparation of asuspension; (b) a preformed suspension of microspheres; and (c) drymicrospheres and a biocompatible solution in separate containers. Thefinal composition of microspheres is injectable through the needle intoa mammal and the microspheres are not capable of being digested oreliminated through said mammal's macrophages or other elements of theimmune system or the lymphatic system.

It should be recognized that both treatments for GERD, urinaryincontinence, urinary reflux disease, and skin deficiencies describedabove can be used in combination with conventional therapies now used totreat these diseases or conditions, i.e., oral diuretics, antacids,suitable drug therapy, cosmetic surgeries and the like. Such combinationtherapy can lead to a faster, safer and more comfortable recovery forthe patient.

As used herein the terms “administered”, “implanted”, or “implantation”are used interchangeably and mean that the material is delivered to thearea of treatment by techniques know to those skilled in the art andappropriate for the disease to be treated. Both invasive andnon-invasive methods may be used for delivery. “Injectable” as used inthe present invention means capable of being administered, delivered orcarried into the body via needle or other similar ways.

As used in the present invention, “microparticles” means polymer orcombinations of polymers made into bodies of various sizes. Themicroparticles can be in any shape, although they are often insubstantially spherical shape, in which case the microparticles arereferred to as “microspheres” or “microbeads.” Before injection or beingcomposed into an injectable composition, the microspheres aresterilized. “Elastic” microparticles or microspheres refers tomicroparticles or microspheres comprise polymers that have elasticproperties. Specific to the present invention, elastic microspheresmeans particles that are flexible enough so that they can be easilyinjected through needles of 18 gauge or smaller, yet the microspheresare not fragile so that they are not broken during the process ofinjection.

The microspheres of the present invention also comprise particles thatare “hydrophilic,” which, as used in the invention, means the particlescan dissolve in, absorb, or mix easily with water or aqueous solution.

“Substantially spherical” generally means a shape that is close to aperfect sphere, which is defined as a volume that presents the lowestexternal surface area. Specifically, “substantially spherical” in thepresent invention means, when viewing any cross-section of the particle,the difference between the average major diameter and the average minordiameter is less than 20%. The surfaces of the microspheres of thepresent invention appear smooth under magnification of up to 1000 times.The microspheres of the present invention may comprise, in addition tothe particles, other materials as described and defined herein.

“Skin wrinkles,” “skin deficiencies,” and “skin contour deficiencies”are used interchangeable in the present invention to refer to skinconditions that are either abnormal or undesirable due to variousinternal or external conditions such as aging, environmental exposure tothe sun and other elements, weight loss, child bearing, disease such asacne and cancer, surgery, wounds, accidents, bites, cuts.

“Dermal augmentation” in the context of the present invention refers toany change of the natural state of a mammal's skin and related areas dueto external acts. The areas that may be changed by dermal augmentationinclude, but not limited to, epidermis, dermis, subcutaneous layer, fat,arrector pill muscle, hair shaft, sweat pore, and sebaceous gland.

“Tissue bulking” in the context of the present invention refers to anychange of the natural state of a mammal's non-dermal soft tissues due toexternal acts or effects. The tissues encompassed by the inventioninclude, but not limited to, muscle tissues, connective tissues, fats,and, nerve tissues. The tissues encompassed by the present invention maybe part of many organs or body parts including, but not limited to, thesphincter, the bladder sphincter and urethra.

“Cell adhesion promoter” in the present invention means any materialthat, because of their presence in or association with the microspheres,promotes or enhances the adhesiveness of cells to the surface of themicrospheres. These materials are often proteins that are bound to thesurface of the microspheres through covalent bonds of the proteins andthe polymers.

“Therapeutic agent” in the present invention refers to any substancethat provides therapeutic effects or biological or physiologicalresponses to the dermal augmentation or tissue bulking procedure. Anexample of therapeutic agent is an anti-inflammation agent that preventsor reduce the effect of inflammations associated dermal augmentation ortissue bulking procedure, an anti-inflammatory agent, an anti-bacterialagent, or an anti-histamine agent.

“Chemical modification” in the present invention means the changes ofchemical properties and characteristics of the microspheres, eitherduring their production process or by way of mixing or contacting themwith various agents or tissues, such that the microspheres have theability to perform, in addition to dermal augmentation or tissuebulking, other functions once injected into the body.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a safe, effective, stable, and longlasting methods of tissue bulking and dermal augmentation, which methodsare useful for the treatment of gastroesophageal reflux disease, urinaryincontinence, urinary reflux disease, and skin deficiencies. Theinvention provides methods of tissue bulking and dermal augmentation byadministrating injectable composition comprising elastic, hydrophilic,non-toxic and substantially spherical microspheres and a biocompatiblecarrier to a mammal in need of treatment for gastroesophageal refluxdisease, urinary incontinence, urinary reflux disease, or skindeficiencies. The methods of the present invention are intended toencompass the following advantages: (1) the injected materials are noteasily displaced within the tissues in which they were originallyinjected, thus the intended dermal augmentation or tissue bulking effectis achieved without repeated administration or causing adverse effectsto the patient, (2) the injected materials are not readily digested,displaced, or eliminated either biochemically or through the immune orlymphatic system, thus the method is more effective and longer lasting,(3) the materials are of sufficient size to be injected through 18 to 26gauge needles, preferably 22 to 24 gauge needles, for tissue bulking or30 gauge or smaller needles for dermal augmentation, thus the method ismore accurate, efficacious and less intrusive to the patient, (4) theinjected particles are flexible, but not fragile, facilitating easyinjection without being broken, thus providing easy and safe injection,and (5) the injected particles are not irregularly shaped and do notclump together, also providing easy and accurate injection. Thesebenefits, whether alone or in combinations, enhance the effectiveness ofthe treatment and are safe, more convenient and comfortable forpatients.

In one embodiment, the present invention uses microparticles,particularly microspheres or microbeads, having a positive charge on itssurface and a cell adhesion promoter and optionally, a cell growthpromoting agent, to treat GERD, urinary incontinence, and skin wrinkles.The microparticles of the invention are preferably used with autologouscells. In other words, the microparticles of the invention are colonizedwith the appropriate cells prior to implantation. This pre-implantation(pre-administration) step has been shown to reduce or eliminateimmunological responses and implantation rejection reactions. Further,the use of non-biodegradable biologically compatible microbeads withpositive charges and autologous cells, whether tissue-specific or not,improves tissue acceptance and overall treatment.

According to the methods of the present invention, treatment of GERD,urinary incontinence, and urinary reflux disease, and skin wrinkles isachievable while avoiding or substantially reducing adverse tissuereactions, including implantation rejection, degradation of particles,resorption, migration and other adverse events. The methods of theinvention also involve increased connective tissue response.

Microbeads or microparticles for use in the present invention are basedon a biocompatible, hydrophilic, substantially spherical, and non-toxicpolymers. The microspheres are injectable through needle of 18 gauge orsmaller and are not capable of being digested or eliminated through themammal's immune or lymphatic system. The polymers may preferably becoated with agents which promote cell adhesion. Living cells may alsoattach to the microparticles forming layered cells therein which linkwith surrounding tissues to enhance long term stability of the beads.

Microparticles intended to be implanted, preferably through injection,in various locations of the body according to the present invention arecomposed of a non-resorbable hydrophilic polymer containing theappropriate material for cell adhesion, and may additionally containradiopaque molecules or other marking agents, to facilitate localizationby radiology prior to or during intervention. The microspheres of thepresent invention comprise elastomers, preferably elastomers selectedfrom the group consisting of acrylic polymers, vinyl alcohol polymers,acrylate polymers, polysaccharides, silicones, or mixtures thereof. Morepreferably, the hydrophilic copolymers usable for this application arethose of the acrylic family such as polyacrylamides and theirderivatives, polyacrylates and their derivatives as well as polyallyland polyvinyl compounds. All of these polymers are crosslinked so as tobe stable and non-resorbable, and can contain within their structureother chemicals displaying particular properties, such as chemotacticeffects, promotion of cell adhesion to cells or tissues, such as cellsof the esophagus wall or the urethra wall, or skin cells, and/or markingagents.

The microparticles for use in the present invention are non-toxic totissues and cells, biocompatible, and adhesive to various cells andtissues at the site of implantation by means of the cell growth theypromote. In addition, these microparticles are non-resorbable andnon-biodegradable, and thus are stable, durable, and will maintain theirgeneral shape and position once implanted at a desired site.

In general, microparticles for use in the present invention may have anyshape, with microparticles which are substantially spherical in shapebeing preferred. Microparticles for use in the present invention mayhave diameters ranging between about 10 μm to about 1000 nm. Preferably,microparticles for use in the present invention which have cells adheredto the surface thereof will have diameters ranging between 50 μm and1000 μm.

For purposes of tissue bulking, the microspheres of the inventionpreferably have diameters ranging from about 10 μm to about 500 nm, morepreferably from about 100 μm to about 300 nm. For purposes of dermalaugmentation, the microspheres preferably have diameters ranging fromabout 10 μm to about 400 μm, preferably from about 50 μm to about 200μm.

Possible variations of the present invention include replacing themicroparticles with any biocompatible, non-toxic non-resorbablepolymeric particles, membrane, fibers or other solid substrates treatedwith an agent promoting cell adhesion. The invention also includeslinear soluble polymers which, after injection, crosslink in situ toconstitute a solid, cell adhesion promoting filling agent. Preparationand/or injection of empty microparticles (microbubbles) that areprepared in advance or are generated in place via the use of appropriatecatheters, are also contemplated in this invention.

The microparticles, or other solid substrates, for use in the presentinvention are flexible, such that they can easily pass into and throughinjection devices and small catheters without being permanently altered,but the microparticles are also resistant to the muscle contractionstress generated during and after the implantation process. They arealso thermally stable which allows for easy, convenient sterilization,and frozen storage.

The microparticles, or other solid substrates, for use in the presentinvention are also stable in suspension which allows the microparticlesor other solid substrates to be formulated and stored in suspension andinjected with different liquids. More specifically, the hydrophilicnature of the microparticles permits placing them in suspension, and inparticular, in the form of sterile and pyrogenic (pyrogen-free)injectable solutions, while avoiding the formation of aggregates oradhesion to the walls of storage containers and implantation devices,such as catheters, syringes, needles, and the like. Preferably, theseinjectable solutions contain microparticles or other solid substratesdistributed approximately in caliber segments ranging between about 10μm and about 2000 μm.

The microparticles of the present invention are both hydrophilic andcationic. The microparticles preferably comprise a copolymer of aneutral hydrophilic monomer, a difunctional monomer, one or moremonomers having a cationic charge, and optionally, a functionalizedmonomer capable of rendering the microparticle detectable. Themicroparticles may also comprise one or more cell adhesion promoters anda marking agent.

The copolymer is preferably a hydrophilic acrylic copolymer whichcomprises in copolymerized form about 25 to about 98% neutralhydrophilic acrylic monomer by weight, about 2 to about 50% difunctionalmonomer by weight and about 0 to about 50% by weight of one or moremonomers having a cationic charge.

By way of example, the copolymers described in French Patent 2,378,808,which is incorporated herein by reference, can be used in accordancewith this invention to prepare the base microparticle copolymer.

As hydrophilic acrylic monomer, acrylamide and its derivatives,methacrylamide and its derivatives or hydroxymethylmethacrylate can beused.

Examples of difunctional monomer, include but are not limited to theN,N′-methylene-bis-acrylamide, N′,N′-diallyltartiamide orglyoxal-bis-acrylamide.

Further, the monomer having a cationic charge, includes but is notlimited to those carrying a tertiary or quaternary amine function,preferably diethylaminoethyl acrylamide, methacrylamidopropyltrimethylammonium or acrylamidoethyl triethylammonium.

In a particularly preferred embodiment, a copolymer comprising about 25to about 98% methacrylamide by weight, about 2 to about 50%N,N-methylene-bis-acrylamide by weight is used.

In one particularly advantageous embodiment of the invention, it ispossible to increase the stability of the microspheres by reticulatingthe adhesion agent. By way of example, in the case of gelatin, thereticulating agent can be chosen among the difunctional chemical agentsreacting on the gelatin amines (e.g., glutaraldehyde, formaldehyde,glyoxal, and the like).

The functionalized monomer is generally obtained by chemical coupling ofthe monomer with a marker, which can be:

a chemical dye, such as Cibacron Blue or Procion Red HE-3B, makingpossible a direct visualization of the microspheres (Boschetti, J.Biochem-Biophys. Meth., 19:21-36 (1989)). Examples of functionalizedmonomer usable for this type of marking N-acryloyl hexamethyleneCibacrone Blue or N-acryloyl hexamethylene Procion Red HE-3B;

a magnetic resonance imaging agent (erbium, gadolinium or magnetite);

a contrasting agent, such as barium or iodine salts, (including forexample acylamino-e-propion-amido)-3-triiodo-2,4,6-benzoic acid, whichcan be prepared under the conditions described by Boschetti et al.(Bull. Soc. Chim., No. 4 France, (1986)). In the case of barium ormagnetite salts, they can be directly introduced in powered form in theinitial monomer solution.

As indicated above it is also possible to mark the microspheres aftertheir synthesis. This can be done, for example, by grafting offluorescent markers derivatives (including for example fluoresceinisothiocyanate (FITC), rhodamine isothiocyanate (RITC) and the like).

Various types of cell adhesion promoters well known in the art may beused in the present invention. In particular, cell adhesion promoterscan be selected from collagen, gelatin, glucosaminoglycans,fibronectins, lectins, polycations (such polylysine, chitosan and thelike), or any other natural or synthetic biological cell adhesion agent.

Preferably, the cell adhesion promoter is present in the microparticle,or other solid substrate, in an amount between about 0.1 to 1 g per mlof settled microparticles.

Microparticles are prepared by suspension polymerization, drop-by-droppolymerization or any other method known to the skilled artisan. Themode of microparticle preparation selected will usually depend upon thedesired characteristics, such as microparticle diameter and chemicalcomposition, for the resulting microparticles. The microparticles of thepresent invention can be made by standard methods of polymerizationdescribed in the art (see, e.g., E. Boschetti, Microspheres forBiochromatography and Biomedical Applications. Part I, Preparation ofMicrobands in: Microspheres, Microencapsulation and Liposomes, JohnWiley & Sons, Arshady R., Ed., vol. 2, p 171-199 (1999), which isincorporated herein by reference). Microspheres are prepared startingfrom an aqueous solution of monomers containing adhesion agents such ascollagen (gelatin is a denatured collagen). The solution is then mixedwith a non-aqueous-compatible solvent to create a suspension ofdroplets, which are then turned into solid gel by polymerization ofmonomers by means of appropriate catalysts. Microspheres are thencollected by filtration or centrifugation and washed.

Cell adhesion promoters or marking agents are introduced on microbeadsby chemical coupling procedures well known in affinity chromatography,referred to by the term “ligand immobilization”. Another method ofintroduction is by diffusion within the gel network that constitutes thebead and then trapping the diffused molecules in place by precipitationor chemical cross-linking. Therapeutic agents, drugs or any other activemolecules that are suitable for transportation by the beads can also beintroduced into the microbeads prior to bead implantation according tothis last method.

The microspheres of the present invention also can be chemicallymodified so that they will “carry” therapeutic effects, vascularizationeffects, anti-vascularization effects, visualization properties,anti-inflammatory effects, anti-bacterial effects, or anti-histamineeffects, or combinations thereof. The chemical modification of themicrospheres of the present invention is made possible by the fact thatthe microspheres comprise particles made of polymers that arecrosslinked so that they can contain chemicals within their structuresthat possess various properties and that they possess uniquecharacteristics associated with surface covalent bonds. The chemicalmodification of the microspheres of the present invention may also occurthrough the interactions between the microspheres and the neighboringcells and tissue after the administration.

The microspheres of the invention can also be obtained by standardmethods of polymerization described in the art such as French Patent2,378,808 and U.S. Pat. No. 5,648,100, each of which is incorporatedherein by reference. In general, the polymerization of monomers insolution is carried out at a temperature ranging between about 0° C. andabout 100° C. and between about 40° C. and about 60° C., in the presenceof a polymerization reaction initiator.

The polymerization initiator is advantageously chosen among the redoxsystems. Notably, it is possible to use combinations of an alkali metalpersulfate with N,N,N′,N′-tetramethylethylenediamine or withdimethylaminopropionitrile, organic peroxides such as benzoyl peroxidesor even 2,2′-azo-bis-isobutyronitrile.

The quantity of initiator used is adapted by one skilled in the art tothe quantity of monomers and the rate of polymerization sought.

Polymerization can be carried out in mass or in emulsion.

In the case of a mass polymerization, the aqueous solution containingthe different dissolved constituents and the initiator undergoespolymerization in an homogeneous medium. This makes it possible toaccess a lump of aqueous gel which can then be separated intomicrospheres, by passing, for example, through the mesh of a screen.

Emulsion or suspension polymerization is the preferred method ofpreparation, since it makes it possible to access directly microspheresof a desired size. It can be conducted as follows: The aqueous solutioncontaining the different dissolved constituents (e.g., differentmonomers, cell adhesion agent), is mixed by stirring, with a liquidorganic phase which is not miscible in water, and optionally in thepresence of an emulsifier. The rate of stirring is adjusted so as toobtain an aqueous phase emulsion in the organic phase forming drops ofdesired diameter. Polymerization is then started off by addition of theinitiator. It is accompanied by an exothermic reaction and itsdevelopment can then be followed by measuring the temperature of thereaction medium.

It is possible to use as organic phase vegetable or mineral oils,certain petroleum distillation products, chlorinated hydrocarbons or amixture of these different solutions. Furthermore, when thepolymerization initiator includes several components (redox system), itis possible to add one of them in the aqueous phase beforeemulsification.

The microspheres thus obtained can then be recovered by cooling,decanting and filtration. They are then separated by size category andwashed to eliminate any trace of secondary product.

The polymerization stage can be followed by a stage of reticulation ofthe cell adhesion agent and possibly by a marking agent stage in thecase of microspheres rendered identifiable by grafting after synthesis.

Microparticles of the present invention which have the specificproperties of cell adhesion and growth promotion can be used directlyfor tissue bulking or dermal augmentation. Moreover, the microparticlesof the present invention can have specific autologous cells grown ontheir surface in vitro, thereby making the microparticles particularlyuseful for tissue bulking or dermal augmentation.

Prior to the present invention, the injection of implantable substancessuspended in a physiological solution into a tissue resulted in theformation of discrete aggregates inside the muscle mass. These discreteaggregates can constitute various amounts of the implanted substancewhich stays together, however, the substance does not become attached toor a part of the tissue itself. This detachment allows the implantedsubstance to move from the original implantation site.

According to the present invention, in order to avoid this problem, themicroparticles may be injected individually and separately, or morepreferably, the surface of the microparticles may be colonized by alayer of cells for better integration and long term stability of theimplant.

Microparticles of the present invention demonstrate superior ability togrow cells on their surfaces. For example, primary muscle cells havebeen successfully adhered to the surface of the microparticles of thepresent invention thereby allowing for a better integration within amuscle tissue. In addition, since the ultimate goal of tissue bulking isto artificially increase tissue mass, preadipocytes have also been usedto colonize the surface of the microparticles prior injection. In thiscase, the preadipocytes have a volume similar to any other regular cell,but after implantation when the preadipocytes are subject to in vivophysiological conditions, they accumulate droplets of fats therebyincreasing the mass of the implant by more than 10% in volume.

The present invention provides a method for causing tissue bulking in amammal. The method comprises administering a composition of elastic,hydrophilic, non-toxic and substantially spherical microspheres in abiocompatible carrier to the mammal. The composition is injectablethrough a needle of about 18 to about 26 gauge and the microspheres arenot capable of being digested or eliminated by macrophage or otherelements of said mammal's lymphatic system. The tissue bulking method ofthe present invention is suitable for the treatment of various tissuedefects including, but not limited to, dental tissue defects, vocal cordtissue defects, or other non-dermal soft tissue defects. The presentmethod is particularly suitable for GERD, urinary incontinence, orurinary reflux disease.

The injection method of the present invention can be carried out by anytype of sterile needles of 18 gauge or smaller and correspondingsyringes or other means for injection, such as a three-way syringe. Theinjection is preferably made into the area that needs tissue bulkingtreatment. The needles, syringes and other means for injection arecommercially available from suppliers such as VWR Scientific Products(West Chester, Pa.), Becton Dickinson, Kendal, and Baxter Healthcare.The size of the syringe and the length of the needle used will dependenton the particular injection based on factors such as the specificdisease or disorders being treated, the location and depth of theinjection, and the volume and specific composition of the injectablesuspension being used. A skilled practitioner will be able to make theselection of syringe and needle based on experience and the teaching ofthe present invention.

The present invention additionally provides a kit for performing dermalaugmentation tissue bulking. The kit comprises an 18 gauge or smallerneedle and a corresponding syringe (both of which are sterile), whereinthe syringe optionally contains a composition comprising biocompatible,elastic, hydrophilic, non-toxic and substantially spherical microspheresand a biocompatible carrier. The composition is injectable through theneedle and the microspheres are not capable of being eliminated bymacrophage or other elements of said mammal's immune or lymphaticsystem. Alternatively, the kit comprises an 18 gauge or smaller needle,a corresponding syringe, and separate containers containing themicrospheres in dried and sterilized form and the biocompatible solvent.The dried sterilized microspheres and the solvent are ready to be mixedfor injection either in their respective containers or in the syringe.These kits are sterile and ready to use. The kits are designed invarious forms based the sizes of the syringe and the needles and thevolume of the injectable composition contained therein, which in turnare based on the specific skin or tissue defects the kits are designedto treat.

According to the present invention, one means of performing tissuebulking in a patient can be described as follows:

-   -   a) Primary cells are extracted from the patient by a simple        biopsy and isolated;    -   b) These cells are grown on the surface of the microparticles        under growth promoting conditions (e.g., possibly using a        nutrient media which contains autologous serum (drawn from the        patient), until confluence);    -   c) The microparticles having the patient's cells grown on the        top are injected into the patient's target tissue to be bulked.

For the treatment of GERD, the microparticles, or other solidsubstrates, are preferably introduced via the esophagus, either byendoscopic delivery or by laparoscopic technique, and are injected intothe walls of the sphincter where the esophagus meets the stomach, i.e.,the lower esophageal sphincter. This decreases the internal lumen of thesphincter muscle thus permitting easier contraction of the muscle withreduced regurgitation of the gastric fluids into the esophagus. Inaddition, this treatment reduces the inflammation of the loweresophagus. The microparticles, or other solid substrates, may also beloaded with X-ray opaque dye or other imaging agents for subsequentX-ray visualization.

In another embodiment, microparticles injected into the sphincter at thejunction of the esophagus and stomach in order to treat GERD may alsoinclude an amount of a drug used to treat GERD, such as H₂ histamineantagonists including cimetidine, ranitidine, famotidine and nizatidine;inhibitors of H⁺,K⁺-ATPase including omeprazole and lansoprazole;antacids including e.g., Al(OH)₃, Mg(OH)₂, and CaCO₃. As with thetreatment of urinary incontinence, urinary reflux disease, and skinwrinkles, the microspheres may also be used with anti-inflammatoryagents, angiogenesis inhibitors, radioactive elements, and antimitoticagents.

Other therapeutic agents to be used in combination with the microspheresor microparticles of the present invention include those for thetreatment of skin disorders, GERD, urinary incontinence and urinaryreflux disease as reported in Goodman & Gilman's The PharmacologicalBasis of Therapeutics, 9th Ed., McGraw-Hill (1996) and The Physicians'sDesk Reference® 2000.

The primary advantages of the method of treating GERD according to thepresent invention over the prior art methods are:

-   -   a) Less invasive effects on the patient compared to surgery;    -   b) More accurate and effective delivery of the microspheres and        therapeutic agents;    -   c) More permanent effects over antacids or other drugs;    -   d) Good biocompatibility with chemotactic effects; and    -   e) Ability to use X-ray visualization or MRI to assist in        follow-up evaluation of the patient.

For the treatment of urinary incontinence and urinary reflux disease,the microparticles or microspheres of the present invention areinjectable through needles of about 18 gauge to about 26 gauge,preferably, 22 to 24 gauge, and are not capable of being eliminatedthrough the lymphatic system. The microparticles are introduced via theurethra and injected into the walls of the bladder sphincter, decreasingthe internal lumen of the sphincter muscle thus permitting easiercontraction of the muscle with reduced likelihood of incontinence. Themicroparticles, or other solid substrate, may also be loaded with X-rayopaque dye, or other imaging agents for subsequent X-ray visualization.

In another embodiment, microparticles injected into the bladdersphincter in order to treat urinary incontinence or urinary refluxdisease may also include an amount of a drug used to treat urinaryincontinence or urinary reflux disease, such as antidiuretics,anticholinergics, oxybutynin and vasopressins.

Injected microparticles can generate some transient adverse reactionssuch as local inflammation, therefore the microparticles can contain orbe injected with anti-inflammatory drugs, such as salicylic acidderivatives including aspirin; para-aminophenol derivatives includingacetaminophen; non-steroidal anti-inflammatory agents includingindomethacin, sulindac, etodolac, tolmetin, diclodfenac, ketorolac,ibuprofen, naproxen, flurbiprofen, ketoprofen, fenoprofen, oxaprozin;anthranilic acids including mefenamic acid, meclofenamic acid; enolicacids such as piroxicam, tenoxicam, phenylbutazone, oxyphenthatrarone;nabumetone; VIOXX (rofecoxib) and CELEBREX (celecoxib). Theseanti-inflammatories are preferably adsorbed on the microparticle'snetwork and released slowly over a short period of time (a few days).The microparticles may also be used to release other specific drugswhich can be incorporated within the microparticle network beforeinjection into the patient. The drug would be released locally at thesite of implantation over a short period of time to improve the overalltreatment.

Incorporation of active molecules, such as drugs, into themicroparticles of the present invention can be accomplished by mixingdry microparticles with solutions of said active molecules or drugs inan aqueous or hydro-organic solution. The microparticles swell byadsorbing the solutions and incorporate the active molecule of interestinto the microparticle network. The active molecules will remain insidethe microparticle due to an active mechanism of adsorption essentiallybased on ion exchange effect. The microparticles by their nature carrycationic groups and have the ability to adsorb anionic molecules, suchas well known anti-inflammatory drugs, and these anionic molecules arethen released slowly upon injection into the patient due to the actionof physiological salt and pH. The ability of various types ofmicroparticles to adsorb drug molecules may be readily determined by theskilled artisan, and is dependent on the amount of cationic monomerspresent in the initial solution from which the microparticles areprepared.

Some of the primary advantages of treating urinary incontinence orurinary reflux disease according to the present invention over prior artmethods are:

-   -   a) More permanent effect than the use of regular viscous        solutions of collagen;    -   b) More accurate and effective delivery of the microspheres and        therapeutic agents;    -   c) Good biocompatibility with chemotactic effect;    -   d) Visualization under X-ray or MRI to assist in follow-up        evaluation; and    -   e) Preventing repeated treatments with resorbable naturally        occurring substances like collagen.

The primary advantages of the method of treating skin wrinkles accordingto the present invention are:

-   -   a) less invasive effects on the patient compared to surgery;    -   b) more accurate and effective delivery of the microspheres and        therapeutic agents;    -   c) more permanent effects than the use of collagen injections;        and    -   d) good biocompatibility with chemotactic effects.

The dermal augmentation method of the present invention comprisesadministering a composition of elastic, hydrophilic, non-toxic andsubstantially spherical microspheres in a biocompatible carrier to amammal in need of such treatment. The composition is injectable throughneedles of about 30 gauge or smaller and the microspheres are notcapable of being digested or eliminated through macrophages or otherelements of the immune system. The injectable composition is preferablya suspension of the microspheres in the biocompatible carrier. Themicrospheres are preferably injected into the mammal's subcutaneouslayer. The microparticles may also include one or more anti-inflammatoryagents.

Suitable for treatment using the dermal augmentation method of thepresent invention are skin contour deficiencies caused by variousconditions including, but not limited to, aging, environmental exposure,weight loss, child bearing, surgery, disease such as acne and cancer, orcombinations thereof. The dermal augmentation method of the presentinvention is particularly suitable for skin contour deficiencies such asfrown lines, worry lines, wrinkles, crow's feet, facial scars,marionette lines, stretch marks, surgical scars, wounds, and cuts andbites due to injury or accidents.

The present invention also provides methods of causing tissue bulking ordermal augmentation by injecting the injectable composition not directlyinto the body, but extracorporeally into organs, components of organs,or tissues prior to their inclusion into the body, organs, or componentsof organs.

The injection of the present invention's method can be preferablycarried out by any type of sterile syringes with needles of about 18 to26 gauge. The size of the syringe and the length of the needle used willdependent on the particular injection based on factors such as thespecific disease or disorders being treated, the location and depth ofthe injection, and the volume and specific composition of the injectablesuspension being used. A skilled practitioner will be able to make theselection of syringe and needle based on experience and the teaching ofthe present invention.

In some embodiments of sphincter bulking, beads are coated and injectedunder physiological conditions into the sphincter. The sphincter volumeincreases proportionally to the amount of injected beads and the lumensize decreases. The beads are progressively and non-reversiblyintegrated within the muscles.

The invention is further defined by reference to the following examplesthat describe in detail the preparation of microparticles for use intissue bulking, and the treatment of skin wrinkles, urinaryincontinence, and GERD. The following examples are illustrative only andshould in no way limit the scope of the present invention. It will beapparent to those skilled in the art that many modifications, both tomaterials and methods, may be practiced without departing from thepurpose and scope of this invention.

EXAMPLES Example 1 Preparation of Irregular Hydrogel Particles withChemotactic Properties

58 grams of sodium chloride and 27 grams of sodium acetate weredissolved at room temperature in 100 ml of demineralized water. To thissolution 400 ml of glycerol were added, the pH was adjusted to 6.0 andmonomers were then dissolved. More specifically to this solution 90 gramof methylolacrylamide, 2 g ofmethacrylamidopropyl-trimethyl-ammonium-chloride hydrochloride and 10gram of N,N′-methylene-bis-acrylamide were added and the mixture wasagitated until complete solubilization. The solution was heated at about70° C. and 100 ml of a solution of gelatin at a concentration of 500mg/ml was added. The total volume of the mixture was then adjusted to1000 ml by addition of demineralized water. Finally 20 ml of 70 mg/mlammonium persulfate aqueous solution and 4 ml ofN,N,N′,N′-tetramethyl-ethylene-diamine was added. The obtained mixturewas stored at 70° C. for about 3 hours until formation of a compactthree-dimensional gel. This gel was totally insoluble in water. It wascut in small pieces and then ground to get very small particles of adimension close to 100-200 μm. The particles were then suspended in 1liter of physiological buffer containing 5% (w/v) glutaraldehyde andwere shaken for two hours. Finally the particles were extensively washedto eliminate unpolymerized material, by-products and salts. To obtainhomogeneous particle size distribution the particle suspension wassieved using an appropriate sieving net.

These particles possess the characteristics desired for tissue celladhesion prior to muscle bulking and include cationic groups andadhesion agents for an effective cell adhesion mechanism.

Example 2 Preparation of Spherical Polyacrylic Hydrogel Gel Particleswith Chemotactic Properties

The solution of monomers prepared as described in Example 1 above waspoured slowly into 1500 ml of stirred and hot paraffin oil (50-70° C.).After a few minutes a suspension/emulsion of liquids was obtained (theaqueous monomer solution was dispersed into oil and forms very smallspherical droplets) and the polymerization occurred in suspension. Themicrodroplets were transformed into microbeads. The solid microbeadswere recovered by centrifugation and suspended in 1 liter ofphysiological buffer containing 5% (w/v) glutaraldehyde and shaken fortwo hours. Finally the particles were extensively washed with water toeliminate completely the oil traces. Organic solvent extraction can beused for a more effective oil removal or an extensive washing in thepresence of traces of nonionic detergents. The obtained microbeads arecalibrated if necessary by sieving through a nylon net and sterilized inan autoclave. These microspheres possess desired characteristics andproperties for cell adhesion prior to muscle bulking.

Example 3 Preparation of Hydrophilic Spherical Polystyrene CopolymerParticles Useful for Tissue Bulking

10 gram of styrene is mixed with 60 ml of toluene. 1 gram ofdivinylbenzene, 1 gram of dimethyl-aminoethyl-methacrylate and 1 gram ofdimethyl-acrylamide are added to the resulting solution. After completesolubilization the monomer solution is mixed with 1% of AIBN(2,2′-azobisisobutyronitrile) as a polymerization catalyst and with 40ml of paraffin oil as a viscosity inducer agent. The mixture is pouredin an agitated water solution containing 0.5% Tween 80. In thissituation there is formation of droplet suspension which turns intosolid microbeads when the temperature is raised to 80-90° C. for threeto five hours. The resulting beads are dried and organic solventsextracted. They are then swollen in an aqueous solution of collagen inphosphate buffer at neutral pH. Embedded collagen is then crosslinkedwith glutaraldehyde as described in Examples 1 and 2. The resultingbeads possess cationic charges to interact with cell tissues andcollagen for cell adhesion, and a chemotactic agent for cell growth andbiocompatibility. They are suitable as tissue bulking agent.

Example 4 Preparation of Hydrophilic Silicone Beads for Cell Adhesionand Tissue Bulking

10 gram of silicone beads of a diameter of 20-300 μm are suspended in 30ml of a solution of hexadecylamine (10 mg/ml) in ethylacetate. Thesuspension is stirred for two hours and 100 ml of ethanol is added. A 1M ammonium sulfate or sodium chloride solution in water is added slowlyuntil a 300 ml suspension is obtained. The amino-containing siliconebeads are then reacted with a butanedioldiglycydylether in alkalineconditions. Epoxy derivatives are thus obtained on which gelatin iscoupled using a method well known in the art. The resulting beads havethe target properties of biocompatibility, hydrophilicity,non-biodegradability and cell adhesion by the presence of cationic aminogroups and of gelatin as a cell growth promoting agent. They aresuitable for tissue bulking in accordance with the present invention.

Example 5 Preparation of Beads for Tissue Bulking Containing AdhesionFactors

Beads prepared according to Example 2 were chemically activated withwell known reagents used in the preparation of affinity chromatographysorbents. Activated beads were then used for the immobilization of celladhesion agents such as fibronectin or vitronectin or laminin. Adhesionagents were dissolved at 1-10 mg/ml in a coupling buffer (100 mMcarbonate or borate buffer pH 8 to 10) and the solution was mixed withthe activated beads. The resulting beads possess the target propertiesof cell adhesion and growth, non-biodegradability and werenon-resorbable. They are suitable for cell adhesion and permanent tissuebulking in accordance with the present invention. Similarly, beadsprepared according to Examples 3 and 4 can also be used.

Example 6 Preparation of Spherical Polyacrylic Hydrogel Particles withChemotactic Properties

Microbeads commercially available under the name SPEC-70 (BioSepra Inc.,Marlborough, Mass.) are polyacrylic polyanionic beads with elasticproperties suitable for tissue bulking applications. However, thesemicrobeads are not chemotactic and do not possess cationic charges.SPEC-70 microbeads are first drained under vacuum to eliminate water andthen suspended in an aqueous solution of 1% chondroitin sulfate sodiumsalt in physiological conditions. Once this compound is absorbed on thebead structure, the beads are drained under vacuum and suspended in anaqueous solution containing 20% polylysine by weight. The suspension isshaken for a few hours and then drained under vacuum and rapidly washedwith distilled water. The beads are then suspended in a solution of 5%butanedioldiglycidylether in ethanol and shaken overnight. Under theseconditions, the polylysine is crosslinked as well as chondroitinsulfate. The resulting modified beads possess properties such ascationic charge for cell adhesion and promoting agents for cell growthsuch as polylysine and chondroitin sulfate.

Example 7 Preparation of Radiopaque Microbeads with ChemotacticProperties for Tissue Bulking

Microbeads from Examples 2 were drained under vacuum and then suspendedin a saturated solution of barium chloride. They were shaken for twohours at room temperature and then drained under vacuum to eliminate theexcess of barium chloride solution. The beads were suspended in asaturated solution of ammonium sulfate and shaken for two additionalhours before elimination of the excess ammonium sulfate by vacuumfiltration. This operation of contact with barium salts and ammoniumsulfate can be repeated several times until the resulting radiopaqueprecipitate inside the beads reaches the desired amount. Resulting beadshave radiopaque properties without having lost their initial desirableproperties for tissue bulking. The microbeads from Examples 3, 4 and 6can be similarly used.

Example 8 Preparation of Radiopaque Microbeads with ChemotacticProperties for Tissue Bulking

Microbeads from Example 6 coated with polylysine are washed extensivelywith distilled water and suspended in a solution of sodium triazoate.The suspension pH is adjusted at about 7 by addition of acetic acid andshaken for several hours. The triazoate which is a radiopaque moleculeis adsorbed tightly to the beads and the remaining reagents areeliminated by washing under vacuum. The resulting beads still possesscell promotion properties and now radiopacity as well.

Example 9 Introduction of Anti-Inflammatory Drugs Inside the BulkingBeads

Microbeads described in the previous Examples may generate localtemporary inflammatory reactions when injected in the target tissue. Toavoid or decrease this phenomenon, the microbeads once coated withautologous cells can be filled with one or more anti-inflammatory drugs.The microbeads are cationic by their nature and can absorb anionic drugsby ion exchange effect.

Prior to injection microbeads are mixed with a 10 mg/mlanti-inflammatory anionic drug solution in sterile physiological saline.The suspension is shaken for several hours, and the beads filled withthe drug are recovered by filtration or centrifugation. The resultinganti-inflammatory containing microbeads may then be used as tissuebulking agents for use in the present invention.

Example 10 In Vitro Pre-Adipocytes Adhesion and Growth on PolymericBeads

In order to assess the ability of polymeric beads from Example 2 toallow adhesion and growth of pre-adipocytes, fresh pre-adipocytes werecollected and isolated from Wistar rat peri-epididymal fat tissue.Pre-adipocytes were then cultured in the presence of above describedmicrobeads at a concentration of about 7.1×10⁵ to about 1.7×10⁶ cells/mlusing the classical protocol for microcarrier culture in vitro. In afirst phase the cells adhere on the bead surface and then they grow tototally cover the bead surface. The total colonization period is about72 hours.

Pre-adipocytes from this type of culture show good growth and specificbiological activity associated with differentiation into adipocytes(accumulation of lipids). Moreover these cells show the presence ofspecific enzymatic markers such as glycerol-3-phosphate-dehydrogenaseand malate dehydrogenase. Microbeads having cells adhered thereto areuseful for tissue bulking for use in the present invention. Thepolymeric beads of Examples 2 to 5 can be similarly assessed.

Example 11 Culture of Pre-Adipocytes and Myocytes on Microbeads In Vitroto Check their Ability of Integrate into an In Vivo Tissue

Preadipocytes and smooth muscle cells were isolated from Wistar ratsaccording to a classical protocol to eliminate most of othercontaminating cells. Separately these cells were cultured in a Petridish in the presence of Dulbecco's Modified Eagle Medium supplementedwith 10% fetal bovine serum. Gelatin-coated cationic microbeads preparedin accordance with Example 2 were added to cells cultured in vitro untilthey covered the surface of the Petri dish. Initial cell seedconcentration was 0.7×10⁶ cells/ml.

Repeated observations showed that cells adhered on the surface ofmicrobeads and further multiplied to cover all the surface of the beads.After 5 to 7 days of culturing, there was formation of a solid networkof beads where cells acted as a binder to consolidate the blocks ofseveral beads. In most cases there were formation of solid nondissociable aggregates comprising beads and cells.

When, after a growing period (generally 5 to 7 days), a differentiatingelement such as 3,3′,5-triiodo-D-thyronine was added to preadipocytes,the preadipocytes started to accumulate fats as micro-droplets withinthe cytoplasm.

Specific staining with 3,3′-dioctadecyloxacarbocyanine perchlorate or2′-[4-hydroxyphenyl]-5-[4-methyl-1-piperazinyl]2,5′-bi-1H-benzimidazoledemonstrated good adhesion of the cells on the bead substrate.

Staining of the cells with red oil at the beginning of thedifferentiating phase evidenced the accumulation of fats inside thecells.

In addition, specific enzymatic reactions of malic enzyme indicatedthat, at the end of the culture, resulting adipocytes were functionallyviable with their major expressed characteristics. This enzyme is notexpressed at the beginning of the culture and appeared simultaneouslywith the accumulation of fats.

Smooth muscle cells were also followed in their proliferation by DNAsynthesis assay; their adhesion on the substrate was followed as perpreadipocyte cells. Myocytes also showed good proliferation as well asadhesion on the beads.

Example 12 In Vitro Myocyte Adhesion and Growth on Polymeric Beads

In order to assess the ability of polymeric beads from Example 2 toallow adhesion and growth of muscle cells, fresh smooth cell myocyteswere collected from rat esophagus according to classical procedures.Cells were then cultured in the presence of above described microbeadsat a concentration of about 10⁶ cells/ml using the classical protocolfor microcarrier culture in vitro. In a first phase the cells adhered onthe bead surface and then they grow until they cover the total beadsurface. The total colonization period was about 72 hours.

Myocytes from this type of culture showed good growth and behavior anddisplayed the specific myosin marker. These microbeads having cellsadhered thereto are useful for tissue bulking in accordance with thepresent invention. The beads from Examples 2 to 5 can be similarlyassessed.

Example 13 Preparation of Injectable Suspension of Cell-MicrobeadParticles for In Vivo Bulking

At the issue of cell culture phase, the cell-bead particles arecollected by filtration and washed extensively with blood serum from thehost where the material is to be implanted. This operation ensures theelimination of foreign material from cell culture. The microbeads arethen suspended in a few ml of autologous serum (a ratio of beads/serumis about 1:1) and are ready to be injected within the tissue to bebulked by means of an appropriate syringe or other injection device.

Example 14 Preparation of Injectable Suspension of Cell-MicrobeadsParticles for In Vivo Bulking

Microbeads described in Example 2 are colonized with rat muscle cellsaccording to Example 10 and conditioned according to Example 13 usingrat serum diluted with physiological saline (50%-50%). The final sterilesuspension of cells anchored on beads (50% of volume is constituted ofbeads and 50% of physiological saline) is injected in the right thighmuscle of a rat. Three months after bead injection the muscle wasobserved in its shape and histologically examined. Muscle volume shouldbe larger than the left thigh muscle upon autopsy. Beads inside themuscle mass should appear surrounded by fibroblastic cells with nospecific adverse inflammatory or necrosis effects.

The embodiments of the present invention described above are intended tobe merely exemplary and those skilled in the art will recognize, or beable to ascertain using no more than routine experimentation, numerousequivalents to the specific procedures described herein. All suchequivalents are considered to be within the scope of the presentinvention and are covered by the following claims.

The contents of all references described herein are hereby incorporatedby reference.

Other embodiments are within the following claims.

What is claimed:
 1. A method for dermal augmentation in a mammalcomprising injecting a composition of non-biodegradable, elastic,hydrophilic, non-toxic and substantially spherical microspheres having adiameter of from about 10 μm to about 400 μm in a biocompatible carrierto said mammal through a needle of about 30 gauge or smaller, whereinthe microspheres comprise one or more of: acrylamide polymers, vinylalcohol polymers, or derivatives and mixtures thereof.
 2. The method ofclaim 1, wherein the composition is a suspension of said microspheres insaid biocompatible carrier.
 3. The method of claim 1, wherein there isno aggregation or clumping of the microspheres prior to or during theadministration.
 4. The method of claim 1, wherein the compositionfurther comprises a therapeutic agent, radio-pacifying agent, contrastmedia, or mixtures thereof.
 5. The method of claim 4, wherein saidtherapeutic agent is bound to the microspheres.
 6. The method of claim1, wherein the microspheres are capable of being chemically modified tohave therapeutic effects, anti-inflammatory effects, anti-bacterialeffects, anti-histamine effects, or combinations thereof.
 7. The methodof claim 6, wherein the chemical modification of the microspheres iscaused by interactions between the microspheres and neighboring tissuesafter administration thereof.
 8. The method of claim 1, wherein theadministration comprises injecting said composition into an area of saidmammal in need of dermal augmentation.
 9. The method of claim 8, whereinthe administration comprises injecting said composition into thesubcutaneous layer.
 10. The method of claim 1, wherein the dermalaugmentation is for treatment of contour deficiencies of said mammal.11. The method of claim 10, wherein the contour deficiencies are causedby aging, environmental exposure, weight loss, child bearing, surgery,disease, or combinations thereof.
 12. The method of claim 11, whereinthe disease is acne, skin cancer, or combination thereof.
 13. The methodof claim 11, wherein the contour deficiencies are one or more of thegroup consisting of frown lines, worry lines, wrinkles, crow's feet,marionette lines, stretch marks, and internal or external scars resultedfrom injury, wound, surgery, bites, cuts, or accident.
 14. The method ofclaim 1, wherein the mammal is human.
 15. The method of claim 1, whereinthe administration comprises injecting said composition extracorporeallyinto organs, components of organs, or tissues prior to their inclusioninto said mammal's body, organs, or components of organs.